Embodiments disclosed herein relate to methods for purifying antibodies, in particular IgG antibodies.
Anion exchange chromatography has been used for the purification of IgG antibodies because it selectively binds acidic contaminants, while IgG binds weakly, not at all, or it is repelled from the anion exchanger surface. This provides a convenient effective means of eliminating acidic contaminants from the antibody. Commonly practiced examples of anion exchange chromatography include bulk addition of positively charged polymers or particles to an IgG-containing sample, passage of the sample through a column packed with anion exchange particles in flow-through or void exclusion mode, or the technique of high performance tangential flow filtration in which the passage of antibody through membrane pores is prevented at very low salt concentrations exclusively by their electrostatic repulsion from positively charged groups on the surface of the membrane. With the exception of anion exchange on particle-packed columns operated in void exclusion mode (R. Nian et al, J. Chromatogr. A 1282 (2013) 127-132), all anion exchange methods require that the sample first be equilibrated to chemical conditions suitable for the binding of contaminants. This restricts the applicability of anion exchange because it means that a sample coming from a previous fractionation step must be buffer exchanged before being applied to an anion exchanger so that the conditions are appropriate for practicing the technique. The even more restrictive option is that the previous fractionation step itself must be selected so that the processed IgG is already provided under conditions suitable for application to an anion exchanger at the completion of that fractionation step. These restrictions particularly burden purification process sequences where an IgG antibody is resident in high salt conditions, such as following a cation exchange chromatography step, or multimodal (cation exchange-hydrophobic interaction, or hydroxyapatite step), or salt precipitation step, or where salt has been added to a sample for any reason.
Methods have been described for processing IgM-containing cell culture harvests that particularly remove chromatin catabolites (Gan et al J. Chromatogr. A, 1291 (2013) 33-40). These methods particularly describe the use of the DNA intercalating compound ethacridine for clarifying an IgG-containing cell culture harvest, under roughly physiological conditions.
Partial purification of monoclonal IgG antibodies by contaminant co-precipitation with caprylic acid (octanoic acid) has been disclosed (Chantuin, A., et al, Arch. Biochem. Biophys. 89 (1960) 218-220). The fatty acid binds to all proteins but selectively precipitates non-IgG contaminants (Gagnon, P., Purification Tools for Monoclonal Antibodies, 1996, Validated Biosystems, Tucson; Morais, V., et al, Biotechnol. Appl. Biochem., 59 (2012) 50-54). Process development guidelines for application to cell-free cell culture harvests have been indicated (Gagnon supra). Application of caprylic acid to cell-containing cell culture harvests has been described (Brodsky et al Biotechnol. Bioeng. 109 (2012) 2589-2598). The technique has the unfortunate feature of co-producing a turbid, sticky, electronegative haze that interferes with further purification (Gagnon supra; Brodsky et al supra).
Allantoin is an FDA-approved inflammatory agent used widely in over-the-counter healthcare products. It is known to remove endotoxin from protein solutions, including from solutions of IgG (V. Vagenende et al, ACS. Appl. Mater. Interfaces, 22 (2013) 4472-4478; V. Vagenende et al, J. Chromatogr. A 1310 (2013) 15-20).